Immobilized pH Gradients IPGs

A variety of precast IPG gels is commercially available from electrophoresis suppliers, and this represents another significant advantage over carrier ampholyte 

The high-resolution and separation stability afforded by isoelectric focusing on immobilized pH gradient gels has also been exploited in 2D electrophoresis methods, where IEF in the first dimension is followed by a second separation method performed in a perpendicular direction. 

Two-dimensional separation techniques are those in which a sample is subjected to two displacement processes that occur orthogonally to each other. The two dimensions should be based upon two completely different separation mechanisms, since identical mechanisms merely yield diagonal separation patterns. The resolving power of 2D techniques can be expressed by Eq. 11.3  

To date, the most powerful combination of electrophoresis techniques employs IEF in the first dimension, resolving on the basis of pi, and SDS-PAGE in the second dimension to separate on the basis of molecular weight. The order is crucial, since the isoelectric point of a protein effectively disappears upon treatment with SDS, which yields a uniformly negative charge/mass ratio. 

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Because the pi of a protein is based on its amino acid sequence, this technique has good resolving power. The resolution can be adjusted further by changing the range of the pH gradient. The use of immobilized pH gradient (IPG) strips has enabled reproducible micropreparative fractionation of protein samples, which is not consistently possible when ampholytes are used in the first dimension (Gorg et al., 2000). 

The majority of proteomic investigations employ two-dimensional gel electrophoresis (2-D) with immobilized pH gradients (IPGs) to separate the proteins in a sample and combine this with mass spectrometry (MS) technologies to identify proteins. 

The technique of immobilized pH gradients (IPG) in isoelectric focusing was first announced to the scientific community at a meeting of the International Electrophoresis Society in Athens, April 1982, as the result of an intensive collaborative effort (Righetti et al., 1983c Bjellqvist et al., 1983 Gorg et al., 1983). It seems extraordinary that in only two and one-half years the method was developed to such an extent as to call for an extensive review. When conventional isoelectric focusing (lEF) was developed in 1960 (Svensson, 1961, 1962), it took a full decade before the scientific community became aware of it. 

The introduction of the immobilized pH gradient (IPG strip) greatly enhances the potential of 2DE (Righetti and Bossi, 1997 Fichmann, 1999 Gorg et al, 2000). This is... 

Righetti, P. G., and Tonani, C. (1991) Immobilized pH gradients (IPG) simulator—An additional step in pH gradient engineering. Electrophoresis 12, 1011-1027. 

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